Comoviruses (CPMV)
Comoviruses are RNA viruses with a bipartite genome. The segments of the comoviral RNA genome are referred to as RNA-1 and RNA-2. RNA-1 encodes the VPg, replicase and protease proteins (Lomonossoff & Shanks, 1983). The replicase is required by the virus for replication of the viral genome. The RNA-2 of the comovirus cowpea mosaic virus (CPMV) encodes a 58K and a 48K protein, as well as two viral coat proteins L and S.
Initiation of translation of the RNA-2 of all comoviruses occurs at two different initiation sites located in the same triplet reading frame, resulting in the synthesis of two carboxy coterminal proteins. This double initiation phenomenon occurs as a result of ‘leaky scanning’ by the ribosomes during translation.
The 5′ terminal start codons (AUGs) in RNA-2 of CPMV occur at positions 115, 161, 512 and 524. The start codons at positions 161 and 512 are in the same triplet reading frame. Initiation at the start codon at position 161 results in the synthesis of a 105K polyprotein while initiation at the start codon at position 512 directs the synthesis of a 95K polyprotein. As the synthesis of both polyproteins is terminated at the same stop codon at position 3299, the 105K and the 95K proteins are carboxy coterminal. The AUG codon at position 524 can serve as an initiator if the AUG at 512 is deleted. However, in the presence of the AUG 512 it does not serve this function and simply codes for the amino acid methionine (Holness et al., 1989; Wellink et al., 1993). The start codon at position 115 is not essential for virus replication (Wellink at al., 1993).
The 105K and 95K proteins encoded by CPMV RNA-2 genome segment are primary translation products which are subsequently cleaved by the RNA1-encoded proteolytic activity to yield either the 58K or the 48K protein, depending on whether it is the 105K or 95K polyprotein that is being processed, and the two viral coat proteins, L and S. Initiation of translation at the start codon at position 512 in CPMV is more efficient than initiation at position 161, resulting in the production of more 95K polyprotein than 105K polyprotein.
The start codon at position 115 in CPMV RNA-2 lies upstream of the initiation sites at positions 161 and 512 and is in a different reading frame. As this start codon is in-phase with a stop codon at position 175, initiation at this site could result in the production of a 20 amino acid peptide. However, production of such a peptide has not been detected to date.
Necessity of Maintaining the Frame between AUGs
Mutagenesis experiments have shown that maintenance of the frame between the initiation sites at positions 161 and 512 in CPMV RNA-2 is essential for efficient replication of RNA-2 by the RNA-1-encoded replicase (Holness et al., 1989; van Bokhoven et al., 1993; Rohll et al., 1993; Wellink et al., 1993). This requirement restricts the length of sequences which can be inserted upstream of the 512 start codon in expression vectors based on CPMV RNA-2 (see below), making the cloning of foreign genes into such vectors more difficult than would be ideal. For example it precludes the use of polylinkers as their use will often alter the open reading frame (ORF) between these initiation sites.
CPMV Vectors
CPMV has served as the basis for the development of vector systems suitable for the production of heterologous polypeptides in plants (Liu et al., 2005; Sainsbury et al., 2007). These systems are based on the modification of RNA-2 but differ in whether full-length or deleted versions are used. In both cases, however, replication of the modified RNA-2 is achieved by co-inoculation with RNA-1. Expression systems based on a full-length version of RNA-2 involve the fusion of the foreign protein to the C-terminus of the RNA-2-derived polyproteins. Release of the N-terminal polypeptide is mediated by the action of the 2A catalytic peptide sequence from foot-and-mouth-disease virus (Gopinath et al., 2000). The resulting RNA-2 molecules are capable of spreading both within and between plants. This strategy has been used to express a number of recombinant proteins, such as the Hepatitis B core antigen (HBcAg) and Small Immune Proteins (SIPs), in cowpea plants (Mechtcheriakova et al., 2006; Monger et al., 2006; Alamillo at al., 2006). Though successful, the use of a full-length viral vector has disadvantages in terms of size constraints of inserted sequences and concerns about biocontainment.
To address these, a system based on a deleted version of CPMV RNA-2 has recently been developed (Cañizares et al., 2006). In this system the region of RNA-2 encoding the movement protein and both coat proteins has been removed. However, the deleted molecules still possess the cis-acting sequences necessary for replication by the RNA-1-encoded replicase and thus high levels of gene amplification are maintained without the concomitant possibility of the modified virus contaminating the environment. With the inclusion of a suppressor of gene silencing, such as HcPro from PVY, (Brigneti et al., 1998) in the inoculum in addition to RNA-1, the deleted CPMV vector can be used as a transient expression system (WO/2007/135480) Bipartite System, Method And Composition For The Constitutive And Inducible Expression Of High Levels Of Foreign Proteins In Plants; also Sainsbury et al., 2009). However, in contrast to the situation with a vector based on full-length RNA-2, replication is restricted to inoculated leaves. These CPMV vectors have been used to express multi-chain complexes consisting of a single type of polypeptide.
Multiple copies of vectors based on either full-length or deleted versions of CPMV RNA-2 have also been shown to be suitable for the production of heteromeric proteins in plants (Sainsbury at al., 2008). Co-infiltration of two full-length RNA-2 constructs containing different marker genes into Nicotiana benthamiana in the presence of RNA-1 has been used to show that two foreign proteins can be efficiently expressed within the same cell in inoculated tissue. Furthermore, the proteins can be co-localised to the same sub-cellular compartments, which is an essential prerequisite for heteromer formation.
The suitability of different CPMV RNA-2 vectors for the expression of heteromeric proteins in plants has also been investigated. Insertion of the heavy and light chains of an IgG into full-length and deleted versions of RNA-2 showed that both approaches led to the accumulation of full-size IgG molecules in the inoculated tissue but that the levels were significantly higher when deleted RNA-2 vectors were used. The ability of full-length RNA-2 constructs to spread systemically therefore seems to be irrelevant to the production of heteromeric proteins and the use of deleted versions of RNA-2 is clearly advantageous, especially as they also offer the benefit of biocontainment.
Thus, known CPMV based vector systems represent useful tools for the expression of a heterologous gene encoding a protein of interest in plants. However, there is still a need in the art for optimised vector systems which improve, for example, the yield of the heterologous proteins expressed and the ease of use of the vector.